# A Study on the Stability and Carbohydrate Metabolic Traits of Starter Cultures in Response to Continuous Subculturing

## TL;DR

This study examines how starter cultures used in fermentation maintain stability and metabolic traits over many generations of subculturing.

## Contribution

The study provides new insights into the physiological and genetic stability of key starter cultures during long-term subculturing.

## Key findings

- Strains showed intact cellular morphology and improved growth and fermentation performance after 2000 generations.
- S. thermophilus mainly used mono- and disaccharides, while L. bulgaricus metabolized diverse substrates and effectively catabolized ribose.
- All strains exhibited genomic stability with fewer than 21 SNPs per isolate.

## Abstract

The industrial application of starter cultures requires stable physiological and genetic performance. In this study, Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were continuously subcultured. Physiological stability was assessed through colony morphology, fermentation activity, and growth profiling. Genetic stability was evaluated through comparative genomics of carbohydrate metabolism networks and single-nucleotide polymorphism (SNP) analysis. The results showed that after 2000 generations, the cellular morphology of the strains remained intact. Additionally, the strains exhibited enhanced growth performance and fermentation capability. The Gompertz model revealed that adapted S. thermophilus A37 and L. bulgaricus B29 exhibited shortened lag phases, increased maximum specific growth rates, and high stationary-phase cell densities. Phenotypic microarray and comparative genomics revealed that S. thermophilus mainly used mono- and disaccharides, with impaired ribose metabolism due to the absence of the rbsk gene in the pentose phosphate pathway. In contrast, L. bulgaricus metabolized diverse oligosaccharides, sugar alcohols, and plant-derived substrates. Additionally, it effectively catabolized ribose through the phosphoketolase pathway and possessed a trehalose degradation cluster. All strains exhibited genomic stability, with SNPs revealing fewer than 21 variations per isolate. This study provides an important theoretical foundation for evaluating the stability of fermentation starter cultures.